We examined genetic diversity within- and among-populations of speckled dace (Rhinichthys osculus) in five major drainage systems in the state of Oregon in western North America. Analysis of sequence variation in a 670-bp segment of the mitochondrial cytochrome b gene revealed deep divergence among basins and high genetic diversity within basins. Application of a molecular clock indicated that the divergence time among basins reflects vicariant events during the late Miocene to early Pliocene. The high levels of genetic diversity observed within basins is likely due to large historic population sizes, in particular, within the Klamath Basin. Two highly divergent mtDNA lineages were found to co-occur in populations in the Klamath Basin. This result may be indicative of a complex history of isolation and reconnection in this basin and/or multiple colonization events. Based on the observed level of mtDNA divergence these lineages may represent two reproductively isolated sympatric taxa. We recommend that major basins be regarded as distinct ESUs based on high levels of subdivision, deep divergences, and reciprocal monophyly among basins.